Signaling, transcriptional, and post-transcriptional events regulate cardiac cell fate decisions during early cardiogenesis. Disruption of such events can lead to congenital heart malformations. In particular, human mutations in transcription factors, such as GATA4, TBX5, NKX2-5 and NOTCHI, result in heart disease in children. Embryonic pathways are reactivated under stress in adult hearts, with GATA4 and MEF2C playing central roles in the transcriptional response during cardiac hypertrophy. Recent studies highlight the importance of protein-protein interactions (PPI) in dictating the transcriptional output of DNA binding transcription factors. However, the complex PPIs that titrate effects of cardiac transcription factors have not been systematically explored. During the previous funding period of this PPG, our discoveries focused on PPIs between members of the Notch and Wnt signaling pathways during cardiac development. In addition, we reported that a combination of cardiac developmental transcription factors, including Gata4, Mef2c and Tbx5, could reprogram non-muscle cells into new cardiomyocyte-like cells in the adult, resulting in cardiac regeneration after injury; the addition of Hand2 further improves reprogramming. Project 1 of this PPG renewal application will test the hypothesis that Gata4, Mef2c, Tbx5, and Hand2 have complex interactions with one another and other key factors to regulate the transcriptional output during cardiac differentiation and cardiac reprogramming. The specific alms are (1) to develop a comprehensive map of the Interactome involving Gata4, Mef2c, Tbx5 and Hand2 during cardiac differentiation of mouse embryonic stem (ES) cells into cardiomyocytes; (2) determine the central dependency of interactomes on Gata4, Tbx5 and Hand2, and the consequences of disease-causing missense mutations In GATA4 on the interactome; and (3) determine the functional consequences of PPIs involving Gata4, Mef2c, Tbx5 and Hand2 on specific genomic loci and integrate PPIs with genome occupancy of the transcription factors during cardiac differentiation and reprogramming. This project will reveal mechanisms underlying cardiac gene regulation and will provide potential points of intervention to positively or negatively titrate transcriptional activity.